Naturally isolated cyclic pentapeptide Longicalycinin A, which showed cytotoxicity to Hep G2 cancer cell line with an IC50 value 13.52 μg/mL, has been successfully synthesized by solid-phase methodology with Fmoc/t-Bu protecting schemes via solution-phase macrocyclization. 2-chlorotrityl chloride resin was used as solid support. Solution phase macrocyclization of linear pentapeptide precursor was carried out by two different routes with mild and severe acidic conditions correspondingly and found that percentage yield of Longicalycinin A under mild acidic condition (33%) was better than severe acidic conditions (13.7%). In addition, the cytotoxicity against Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) cell lines with CTC50 values were found to be 2.62 and 6.17 µM respectively.